幼猪肺泡上皮和血管内皮祖细胞分离培养技术探讨
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摘要
研究背景
肺泡Ⅱ型上皮细胞(Alveolar epithelial typeⅡcells, AECⅡ)是肺泡上皮的祖细胞,能够变为Ⅰ型肺泡上皮细胞参与肺损伤修复,分泌肺泡表面活性物质(Pulmonary surfactant, PS)维持肺泡稳定性、防止肺泡萎陷、保持肺的顺应性,能够将肺泡上皮顶端膜面的钠、水转运至肺间质,并参加了肺对各种吸入有害物质及微生物的天然免疫等。AECⅡ仅占肺部细胞的15%,对肺组织或肺混合细胞的培养难以明确AECⅡ的具体功能。目前尚无具有全部AECⅡ功能的细胞系,AECⅡ的原代培养成为解决这一问题的重要手段。以往研究大多是对成年大鼠、小鼠和兔AECⅡ的分离培养研究,也有少量新生动物细胞分离培养报道,但细胞产量均较低,此外由于小动物生长发育周期及生理病理过程短,生理及解剖特点也不利于长期动物实验研究,对于人新生儿至婴幼儿期的生理和病理生理学研究适用性有限。本研究的目的是建立新生猪AECⅡ分离、纯化及鉴定方法,为AECⅡ的生物学特性研究及与AECⅡ有关的在体动物实验研究奠定基础。
目的
1、建立新生猪AECⅡ的分离、纯化及鉴定方法；
2、观察AECⅡ的体外生长变化特点,明确其体外实验的最佳时间。
方法
1、取体重1000-1300 g足月新生猪肺,经气道灌入0.1%胰酶及不同浓度弹力蛋白酶与0.1%胰酶的混合酶溶液(40 u／ml elatase／0.1% trypsin、30 u／mlelatase/0.1% trypsin、20 u/ml elatase/0.1% trypsin) 37℃、20 min水浴消化,收集细胞、计数细胞产量及活力；
2、采用percoll非连续密度梯度离心及免疫粘附法纯化(即panning法)细胞,计数细胞产量及活力；
3、采用透射电镜法、碱性磷酸酯酶染色法鉴定AECⅡ；
4、将细胞以4x105 cells／cm2接种于组织培养板,用DMEM低糖培养基(含10%胎牛血清、100 u／ml青霉素、0.1 g／ml链霉素)进行培养,每24 h换液,观察细胞生长变化情况。
5、在相同的消化条件下：0.1%胰酶、37℃、20 min水浴消化条件下,对比研究免疫粘附法、percoll非连续密度梯度离心法纯化细胞后所获得大鼠、新生猪AECⅡ的产量、活力及纯度的差别。
结果
1、新生猪肺组织细胞的消化分离：30 u／ml弹力蛋白酶／0.1%胰酶消化肺组织后细胞产量(5.35±0.54)x106,而20 u／ml弹力蛋白酶／0.1%胰酶消化细胞产量为(3.16±0.94)x106,40 u／ml弹力蛋白酶／0.1%胰酶消化细胞产量为(3.09±0.86)x106,0.1%胰酶消化细胞产量为(2.76±0.65)x10+,30 u／ml弹力蛋白酶／0.1%胰酶消化细胞产量显著高于其他3组(P<0.01)；
2、新生猪肺AECⅡ的纯化：免疫粘附法纯化后的细胞产量为(37.97±27.98)x106,percoll非连续密度梯度离心法纯化细胞的产量(11.07±10.59)x106,前者明显高于后者；
3、新生猪肺AECⅡ的培养：原代培养24 h,AECⅡ开始贴壁,细胞呈圆形、多角形,呈岛状分布。培养至48 h,细胞形态一致,为多角形。第3-4天细胞平展,连接成细胞单层,胞浆内有大量反差明显的小颗粒,细胞核明显。第5-7天,细胞内颗粒逐渐减少,胞浆空泡,细胞体积增大,边缘模糊。AECⅡ在原代培养24-96 h处于最佳状态,此阶段适合作体外实验研究。
4、在相同消化、分离及纯化方法条件下,新生猪肺AECⅡ的纯化产量明显高于大鼠AECⅡ纯化产量。大鼠AECⅡ的percoll法纯化细胞产量显著高于免疫黏附法。新生猪AECⅡ的纯化,免疫粘附法产量则明显高于percoll法。纯化后大鼠AECⅡ的阳性率近90%,而新生猪AECⅡ的阳性率仅为70%左右。
结论
1、新生猪AECⅡ消化分离的最佳消化条件是：30 u／ml弹力蛋白酶／0.1%胰酶联合使用,37℃、20 min消化；免疫粘附法纯化新生猪AECⅡ优于percoll法；AKP染色法是简单、实用的AECⅡ鉴定方法,细胞染色阳性率与电镜鉴定结果一致,可用于新生猪AECⅡ的鉴定。
2、AECⅡ体外研究的最佳时间为原代培养的第24-96 h。
3、在消化条件及纯化方法相同的情况下,新生猪肺AECⅡ的纯化产量明显高于大鼠AECⅡ纯化产量,AECⅡ纯度可达70%左右,可用于进一步的体外实验研究。
研究背景
内皮祖细胞(Endothelial progenitor cells，EPC)是循环中骨髓来源的祖细胞之一，能分化为成熟血管内皮细胞，又称为血管内皮细胞的前体细胞。正常生理状态下，外周血中EPC数量很少，缺血、血管损伤等因素可以使外周血中EPC数量增加并参与了损伤血管的修复过程。对急性肺损伤(Acute lung injury,ALI)的研究表明，炎症反应抑制剂、炎症因子特异性阻断剂等治疗效果并不佳。目前干细胞治疗在组织修复和重建领域中备受关注，EPC很可能是极具前景的急性肺损伤治疗方法之一。本研究的目的是建立幼猪EPC分离培养方法，为EPC对急性肺损伤修复作用机制研究奠定基础。
目的
建立幼猪外周血EPC体外分离、纯化及鉴定方法，探讨其体外培养条件及生长变化特点。为进一步的EPC生物学特性研究及内皮祖细胞的急性肺损伤修复研究奠定基础。
方法
取幼猪外周血10-20 ml，用密度梯度离心法分离出血中单个核细胞，贴壁选择法纯化EPC，EGM-2MV培养基(含5％FBS，Hydrocortisone 0．4μl/ml，hFGF-B4μl／ml，VEGF 1 ttl／ml，IGF-11μl／ml，Ascorbic acidμl／ml，hEGF 1μl/ml，GA-10001μl/tl／m1)培养细胞，观察细胞生长变化特点。
结果
1．使用淋巴细胞分离液(LTS 1110)，采用密度梯度离心法分离猪外周血单个核细胞的方法稳定。
2．幼猪外周血单个核细胞贴壁选择法纯化后培养观察：细胞贴壁较为缓慢，贴壁细胞大小较为均一、圆形为主。培养7天见较多长梭形细胞，形态相对单
一、触角少。长梭形细胞可逐渐增多，培养至3周左右时见长梭形细胞相互连接呈管腔样结构分布。
3。透射电镜观察管腔样结构分布细胞：可见细胞间有细胞连接。
结论
使用淋巴细胞分离液(LTS 1110)，密度梯度离心法分离幼猪外周血单个核细胞，贴壁选择纯化的方法，可用于与EPC有关的进一步的实验研究中。
Background Alveolar epithelial typeⅡcells (AECⅡ) are progenitor cells for alveolar epithelium and responsible for reformation of alveolar inner lining after impairment of the very sensitive typeⅠepithelial cells (AECⅠ). It synthesizes and secretes pulmonary surfactant (PS) to generate low surface tension at air-liquid interface, preventing collapse and maintaining compliance of lung during cyclic expansion, facilitating transportation of sodium and fluid from the apical surface into the interstitium and plays an important role in innate immune to various noxious substances and organisms from ambient air. As AECⅡcomprises only 15% of all lung cells, it is difficult to attribute specific functions to these cells from the whole lung or mixed cell culture. At present, there is no passaged line that exhibits the full range of known AECⅡfunctions, and the primary culture of AECⅡbecomes mainstay in understanding cell biology and pathobiology. Most of previous studies are based on the small sized, adult animals such as rat, mouse and rabbit, or their newborns, but the yield of cell quantity is small, and the conclusions are not readily applicable in human neonate and infant. In addition, small animals can not be used for long term experiment for the limited developmental lifespan and feature of physiology and anatomy. The aim of this study was to establish a method of isolating and purifying AECⅡfrom newborn piglet lungs in order to provide a foundation for the biological and pathobiological study of acute and chronic lung injury as well as assessment of efficacies and mechanisms of new international strategies and therapeutics relevant in animal studies in vivo.
Objectives
1. To establish methods of isolating, purifying and identifying AECⅡof newborn piglet.
2. To characterize the growing feature of AECⅡduring primary culture for determination of the optimal timing of in vitro study.
Methods
1. The lung of newborn piglet weighing 1000-1300 g was dissociated with different concentration (0.1% trypsin,40 u/ml elatase/0.1% trypsin,30 u/ml elatase/0.1% trypsin,20 u/ml elatase/0.1% trypsin) by instilling the solution into the alveoli through airway and incubated for 20 min at 37℃. The mixed cell were collected, counted and stained with trypan blue for living cell rate calculation.
2. The mixed cells were purified by percoll gradient centrifugation and immune adherence respectively and counted, stained with trypan blue for living cell rate calculation.
3. AECⅡcell were identified by transmission electron microscope observation and alkaline phosphatase staining.
4. The cells were seeded in 6-well plates at a density of 4×105 cells/cm2 and grown in DMEM supplemented with 10% fetal bovine serum (FBS) in 5% CO2-95% air atmosphere at 37℃. After 24 hours culture, unattached cells were removed and the medium was changed for the first time. Since then, medium was changed and cells were observed under inverted microscope every 24 h.
5. The lung tissue of rat and newborn piglet was dissociated with trypsin in the concentration of 0.1% and incubated at 37℃for 20 min. The mixed cells were purified by percoll gradient centrifugation and immune adherence respectively. The numbers, purity and viability of purified cells were compared of these two animals.
Results
1. The cell numbers obtained with 30 u/ml elatase and 0.1% trypsin in combination were significantly larger than the other three groups (40 u/ml elatase and 0.1% trypsin in combination,20 u/ml elatase and 0.1% trypsin in combination,0.1% trypsin) (P<0.01).
2. The number of purified cells obtained using immune adherence was 37.97±27.98 x106 and much larger than those obtained by percoll gradient centrifugation (11.07±10.59x106).
3. AECⅡadhered after 24 h of primary culture, the cells were round and polygon and formed islet like structure. At 48 h of culture, the cell became uniformly polygon shaped. On day 3-4 in culture, the cells expanded and formed confluent monolayer. There were fine particles in intra-cytoplasm and the nucleus were seen clearly. On days 5-7 in culture, fine particles decreased, vacuolus appeared in intra-cytoplasm, the cells expanded and the edge of cells can not be seen clearly. During the 24-96 h in culture, the AECⅡwere in good condition and optimal for in vitro study.
4. AEC II cells obtained from newborn piglet was significantly larger than those from rat under the same isolating and purifying condition. AEC II cells obtained from rat by percoll gradient centrifugation were significantly larger than those by immune adherence. Alveolar type II cells obtained from newborn piglet by immune adherence were much larger than those obtained by percoll gradient centrifugation. The positive alveolar type II cells obtained from rat lung assessed by alkaline phosphatase (AKP) was close to 90%, and it was only about 70% of newborn piglet.
Conclusions
1. The optimal condition for isolating AECⅡof newborn piglet was that to incubate the lung tissue with proteolytic enzymes of 30 u/ml elatase and 0.1% trypsin in combination for 20 min at 37℃. The immune adherence method is much better than percoll gradient centrifugation to purify the mixed cells obtained from newborn piglet lung. Alkaline phosphatase staining is a simple and practical method for identifying AECⅡ. The positive cells assessing by alkaline phosphatase staining is consistent with transmission electron microscope observation of alveolar epithelial typeⅡcell. Alkaline phosphatase staining can be used for identifying alveolar typeⅡcells of newborn piglet.
2. The optimal time for AECⅡin vitro study is during the 24-96 h in primary culture.
3. AECⅡobtained from newborn piglet lung is much larger than those obtained from rat under the same isolating and purifying condition, the positive cells assessing by alkaline phosphatase (AKP) is about 70% and may be used for further study in virto.
Background EPC (endothelial progenitor cells) is one of the progenitors originated from the bone marrow. It can differentiate into mature vascular endothelial cells and its number is very few in peripheral blood in physiological condition but increased substantially when affected by ischemia, vessel injury or other stimulations, thereby participating vascular repair process. Results from acute lung injury (ALI) study indicate that therapeutic efficacy of inhibitor and blockade to inflammatory factor is not satisfactory in clinical practice, especially when the lungs are severely impaired. At present, stem cell therapy receives much attention in the field of tissue reparation and reconstitution, and EPC is regarded as a perspective therapeutic method. The aim of this study was to establish a method to isolate and culture EPC, and to provide a foundation for studies in understanding the reparation mechanism of EPC in ALI and its pathology.
Objectives
To establish methods of isolating and purifying EPC from young piglet so that enables conducting study in the reparative mechanism ALI.Methods
1. The mononuclear cells were isolated from 10-20 ml of porcine peripheral blood with porcine lymphocyte separating medium (LTS1110) by density gradient centrifugation.
2. The mononuclear cells were purified by differential adhering method.
3. The purified cells were seeded in 6-well plates and grown in EGM-2 medium supplemented with 5% fetal bovine serum (FBS), 0.4 ul/ml hydrocortisone, 4 ul/ml hFGF-B, 1 ul/ml VEGF, 1μl/ml IGF-1, 1μl/ml ascorbic acid, 1μl/ml hEGF, 1μl/ml GA-1000. The cells were cultured in 5% CO_2-95% air atmosphere at 37°C. On day 4 in culture, unattached cells were removed and the medium was changed for the first time. Since then, medium was changed and cells wereobserved with inverted microscope every 3 days.
Results
1. The method is stable to isolate the mononuclear cells of porcine peripheral blood with lymphocyte separating medium (LTS 1110) by density gradient centrifugation.
2. The purified cells adhered very slowly, uniform in size and mainly round shaped. Spindle shaped cells were seen in homogeneous form on day 7 in culture with few tentacle. The spindle shaped cells proliferated and formed lumen-like structure on day about 3 weeks in culture.
3. The cell junction was seen under transmission electron microscope between the connected cells forming lumen-like structure.
Conclusions
The method of isolating the mononuclear cells of porcine peripheral blood with porcine lymphocyte separating medium (LTS 1110) by density gradient centrifugation and then purified by differential adhering method, incubated in EGM-2MV medium. This procedure may be used in further study based on isolation and culture of piglet EPC.
肺泡Ⅱ型上皮细胞(Alveolar epithelial typeⅡcells, AECⅡ)是肺泡上皮的祖细胞,能够变为Ⅰ型肺泡上皮细胞参与肺损伤修复,分泌肺泡表面活性物质(Pulmonary surfactant, PS)维持肺泡稳定性、防止肺泡萎陷、保持肺的顺应性,能够将肺泡上皮顶端膜面的钠、水转运至肺间质,并参加了肺对各种吸入有害物质及微生物的天然免疫等。AECⅡ仅占肺部细胞的15%,对肺组织或肺混合细胞的培养难以明确AECⅡ的具体功能。目前尚无具有全部AECⅡ功能的细胞系,AECⅡ的原代培养成为解决这一问题的重要手段。以往研究大多是对成年大鼠、小鼠和兔AECⅡ的分离培养研究,也有少量新生动物细胞分离培养报道,但细胞产量均较低,此外由于小动物生长发育周期及生理病理过程短,生理及解剖特点也不利于长期动物实验研究,对于人新生儿至婴幼儿期的生理和病理生理学研究适用性有限。本研究的目的是建立新生猪AECⅡ分离、纯化及鉴定方法,为AECⅡ的生物学特性研究及与AECⅡ有关的在体动物实验研究奠定基础。
目的
1、建立新生猪AECⅡ的分离、纯化及鉴定方法；
2、观察AECⅡ的体外生长变化特点,明确其体外实验的最佳时间。
方法
1、取体重1000-1300 g足月新生猪肺,经气道灌入0.1%胰酶及不同浓度弹力蛋白酶与0.1%胰酶的混合酶溶液(40 u／ml elatase／0.1% trypsin、30 u／mlelatase/0.1% trypsin、20 u/ml elatase/0.1% trypsin) 37℃、20 min水浴消化,收集细胞、计数细胞产量及活力；
2、采用percoll非连续密度梯度离心及免疫粘附法纯化(即panning法)细胞,计数细胞产量及活力；
3、采用透射电镜法、碱性磷酸酯酶染色法鉴定AECⅡ；
4、将细胞以4x105 cells／cm2接种于组织培养板,用DMEM低糖培养基(含10%胎牛血清、100 u／ml青霉素、0.1 g／ml链霉素)进行培养,每24 h换液,观察细胞生长变化情况。
5、在相同的消化条件下：0.1%胰酶、37℃、20 min水浴消化条件下,对比研究免疫粘附法、percoll非连续密度梯度离心法纯化细胞后所获得大鼠、新生猪AECⅡ的产量、活力及纯度的差别。
结果
1、新生猪肺组织细胞的消化分离：30 u／ml弹力蛋白酶／0.1%胰酶消化肺组织后细胞产量(5.35±0.54)x106,而20 u／ml弹力蛋白酶／0.1%胰酶消化细胞产量为(3.16±0.94)x106,40 u／ml弹力蛋白酶／0.1%胰酶消化细胞产量为(3.09±0.86)x106,0.1%胰酶消化细胞产量为(2.76±0.65)x10+,30 u／ml弹力蛋白酶／0.1%胰酶消化细胞产量显著高于其他3组(P<0.01)；
2、新生猪肺AECⅡ的纯化：免疫粘附法纯化后的细胞产量为(37.97±27.98)x106,percoll非连续密度梯度离心法纯化细胞的产量(11.07±10.59)x106,前者明显高于后者；
3、新生猪肺AECⅡ的培养：原代培养24 h,AECⅡ开始贴壁,细胞呈圆形、多角形,呈岛状分布。培养至48 h,细胞形态一致,为多角形。第3-4天细胞平展,连接成细胞单层,胞浆内有大量反差明显的小颗粒,细胞核明显。第5-7天,细胞内颗粒逐渐减少,胞浆空泡,细胞体积增大,边缘模糊。AECⅡ在原代培养24-96 h处于最佳状态,此阶段适合作体外实验研究。
4、在相同消化、分离及纯化方法条件下,新生猪肺AECⅡ的纯化产量明显高于大鼠AECⅡ纯化产量。大鼠AECⅡ的percoll法纯化细胞产量显著高于免疫黏附法。新生猪AECⅡ的纯化,免疫粘附法产量则明显高于percoll法。纯化后大鼠AECⅡ的阳性率近90%,而新生猪AECⅡ的阳性率仅为70%左右。
结论
1、新生猪AECⅡ消化分离的最佳消化条件是：30 u／ml弹力蛋白酶／0.1%胰酶联合使用,37℃、20 min消化；免疫粘附法纯化新生猪AECⅡ优于percoll法；AKP染色法是简单、实用的AECⅡ鉴定方法,细胞染色阳性率与电镜鉴定结果一致,可用于新生猪AECⅡ的鉴定。
2、AECⅡ体外研究的最佳时间为原代培养的第24-96 h。
3、在消化条件及纯化方法相同的情况下,新生猪肺AECⅡ的纯化产量明显高于大鼠AECⅡ纯化产量,AECⅡ纯度可达70%左右,可用于进一步的体外实验研究。
研究背景
内皮祖细胞(Endothelial progenitor cells，EPC)是循环中骨髓来源的祖细胞之一，能分化为成熟血管内皮细胞，又称为血管内皮细胞的前体细胞。正常生理状态下，外周血中EPC数量很少，缺血、血管损伤等因素可以使外周血中EPC数量增加并参与了损伤血管的修复过程。对急性肺损伤(Acute lung injury,ALI)的研究表明，炎症反应抑制剂、炎症因子特异性阻断剂等治疗效果并不佳。目前干细胞治疗在组织修复和重建领域中备受关注，EPC很可能是极具前景的急性肺损伤治疗方法之一。本研究的目的是建立幼猪EPC分离培养方法，为EPC对急性肺损伤修复作用机制研究奠定基础。
目的
建立幼猪外周血EPC体外分离、纯化及鉴定方法，探讨其体外培养条件及生长变化特点。为进一步的EPC生物学特性研究及内皮祖细胞的急性肺损伤修复研究奠定基础。
方法
取幼猪外周血10-20 ml，用密度梯度离心法分离出血中单个核细胞，贴壁选择法纯化EPC，EGM-2MV培养基(含5％FBS，Hydrocortisone 0．4μl/ml，hFGF-B4μl／ml，VEGF 1 ttl／ml，IGF-11μl／ml，Ascorbic acidμl／ml，hEGF 1μl/ml，GA-10001μl/tl／m1)培养细胞，观察细胞生长变化特点。
结果
1．使用淋巴细胞分离液(LTS 1110)，采用密度梯度离心法分离猪外周血单个核细胞的方法稳定。
2．幼猪外周血单个核细胞贴壁选择法纯化后培养观察：细胞贴壁较为缓慢，贴壁细胞大小较为均一、圆形为主。培养7天见较多长梭形细胞，形态相对单
一、触角少。长梭形细胞可逐渐增多，培养至3周左右时见长梭形细胞相互连接呈管腔样结构分布。
3。透射电镜观察管腔样结构分布细胞：可见细胞间有细胞连接。
结论
使用淋巴细胞分离液(LTS 1110)，密度梯度离心法分离幼猪外周血单个核细胞，贴壁选择纯化的方法，可用于与EPC有关的进一步的实验研究中。
Background Alveolar epithelial typeⅡcells (AECⅡ) are progenitor cells for alveolar epithelium and responsible for reformation of alveolar inner lining after impairment of the very sensitive typeⅠepithelial cells (AECⅠ). It synthesizes and secretes pulmonary surfactant (PS) to generate low surface tension at air-liquid interface, preventing collapse and maintaining compliance of lung during cyclic expansion, facilitating transportation of sodium and fluid from the apical surface into the interstitium and plays an important role in innate immune to various noxious substances and organisms from ambient air. As AECⅡcomprises only 15% of all lung cells, it is difficult to attribute specific functions to these cells from the whole lung or mixed cell culture. At present, there is no passaged line that exhibits the full range of known AECⅡfunctions, and the primary culture of AECⅡbecomes mainstay in understanding cell biology and pathobiology. Most of previous studies are based on the small sized, adult animals such as rat, mouse and rabbit, or their newborns, but the yield of cell quantity is small, and the conclusions are not readily applicable in human neonate and infant. In addition, small animals can not be used for long term experiment for the limited developmental lifespan and feature of physiology and anatomy. The aim of this study was to establish a method of isolating and purifying AECⅡfrom newborn piglet lungs in order to provide a foundation for the biological and pathobiological study of acute and chronic lung injury as well as assessment of efficacies and mechanisms of new international strategies and therapeutics relevant in animal studies in vivo.
Objectives
1. To establish methods of isolating, purifying and identifying AECⅡof newborn piglet.
2. To characterize the growing feature of AECⅡduring primary culture for determination of the optimal timing of in vitro study.
Methods
1. The lung of newborn piglet weighing 1000-1300 g was dissociated with different concentration (0.1% trypsin,40 u/ml elatase/0.1% trypsin,30 u/ml elatase/0.1% trypsin,20 u/ml elatase/0.1% trypsin) by instilling the solution into the alveoli through airway and incubated for 20 min at 37℃. The mixed cell were collected, counted and stained with trypan blue for living cell rate calculation.
2. The mixed cells were purified by percoll gradient centrifugation and immune adherence respectively and counted, stained with trypan blue for living cell rate calculation.
3. AECⅡcell were identified by transmission electron microscope observation and alkaline phosphatase staining.
4. The cells were seeded in 6-well plates at a density of 4×105 cells/cm2 and grown in DMEM supplemented with 10% fetal bovine serum (FBS) in 5% CO2-95% air atmosphere at 37℃. After 24 hours culture, unattached cells were removed and the medium was changed for the first time. Since then, medium was changed and cells were observed under inverted microscope every 24 h.
5. The lung tissue of rat and newborn piglet was dissociated with trypsin in the concentration of 0.1% and incubated at 37℃for 20 min. The mixed cells were purified by percoll gradient centrifugation and immune adherence respectively. The numbers, purity and viability of purified cells were compared of these two animals.
Results
1. The cell numbers obtained with 30 u/ml elatase and 0.1% trypsin in combination were significantly larger than the other three groups (40 u/ml elatase and 0.1% trypsin in combination,20 u/ml elatase and 0.1% trypsin in combination,0.1% trypsin) (P<0.01).
2. The number of purified cells obtained using immune adherence was 37.97±27.98 x106 and much larger than those obtained by percoll gradient centrifugation (11.07±10.59x106).
3. AECⅡadhered after 24 h of primary culture, the cells were round and polygon and formed islet like structure. At 48 h of culture, the cell became uniformly polygon shaped. On day 3-4 in culture, the cells expanded and formed confluent monolayer. There were fine particles in intra-cytoplasm and the nucleus were seen clearly. On days 5-7 in culture, fine particles decreased, vacuolus appeared in intra-cytoplasm, the cells expanded and the edge of cells can not be seen clearly. During the 24-96 h in culture, the AECⅡwere in good condition and optimal for in vitro study.
4. AEC II cells obtained from newborn piglet was significantly larger than those from rat under the same isolating and purifying condition. AEC II cells obtained from rat by percoll gradient centrifugation were significantly larger than those by immune adherence. Alveolar type II cells obtained from newborn piglet by immune adherence were much larger than those obtained by percoll gradient centrifugation. The positive alveolar type II cells obtained from rat lung assessed by alkaline phosphatase (AKP) was close to 90%, and it was only about 70% of newborn piglet.
Conclusions
1. The optimal condition for isolating AECⅡof newborn piglet was that to incubate the lung tissue with proteolytic enzymes of 30 u/ml elatase and 0.1% trypsin in combination for 20 min at 37℃. The immune adherence method is much better than percoll gradient centrifugation to purify the mixed cells obtained from newborn piglet lung. Alkaline phosphatase staining is a simple and practical method for identifying AECⅡ. The positive cells assessing by alkaline phosphatase staining is consistent with transmission electron microscope observation of alveolar epithelial typeⅡcell. Alkaline phosphatase staining can be used for identifying alveolar typeⅡcells of newborn piglet.
2. The optimal time for AECⅡin vitro study is during the 24-96 h in primary culture.
3. AECⅡobtained from newborn piglet lung is much larger than those obtained from rat under the same isolating and purifying condition, the positive cells assessing by alkaline phosphatase (AKP) is about 70% and may be used for further study in virto.
Background EPC (endothelial progenitor cells) is one of the progenitors originated from the bone marrow. It can differentiate into mature vascular endothelial cells and its number is very few in peripheral blood in physiological condition but increased substantially when affected by ischemia, vessel injury or other stimulations, thereby participating vascular repair process. Results from acute lung injury (ALI) study indicate that therapeutic efficacy of inhibitor and blockade to inflammatory factor is not satisfactory in clinical practice, especially when the lungs are severely impaired. At present, stem cell therapy receives much attention in the field of tissue reparation and reconstitution, and EPC is regarded as a perspective therapeutic method. The aim of this study was to establish a method to isolate and culture EPC, and to provide a foundation for studies in understanding the reparation mechanism of EPC in ALI and its pathology.
Objectives
To establish methods of isolating and purifying EPC from young piglet so that enables conducting study in the reparative mechanism ALI.Methods
1. The mononuclear cells were isolated from 10-20 ml of porcine peripheral blood with porcine lymphocyte separating medium (LTS1110) by density gradient centrifugation.
2. The mononuclear cells were purified by differential adhering method.
3. The purified cells were seeded in 6-well plates and grown in EGM-2 medium supplemented with 5% fetal bovine serum (FBS), 0.4 ul/ml hydrocortisone, 4 ul/ml hFGF-B, 1 ul/ml VEGF, 1μl/ml IGF-1, 1μl/ml ascorbic acid, 1μl/ml hEGF, 1μl/ml GA-1000. The cells were cultured in 5% CO_2-95% air atmosphere at 37°C. On day 4 in culture, unattached cells were removed and the medium was changed for the first time. Since then, medium was changed and cells wereobserved with inverted microscope every 3 days.
Results
1. The method is stable to isolate the mononuclear cells of porcine peripheral blood with lymphocyte separating medium (LTS 1110) by density gradient centrifugation.
2. The purified cells adhered very slowly, uniform in size and mainly round shaped. Spindle shaped cells were seen in homogeneous form on day 7 in culture with few tentacle. The spindle shaped cells proliferated and formed lumen-like structure on day about 3 weeks in culture.
3. The cell junction was seen under transmission electron microscope between the connected cells forming lumen-like structure.
Conclusions
The method of isolating the mononuclear cells of porcine peripheral blood with porcine lymphocyte separating medium (LTS 1110) by density gradient centrifugation and then purified by differential adhering method, incubated in EGM-2MV medium. This procedure may be used in further study based on isolation and culture of piglet EPC.
引文
1. Emura M. Stem cells of the respiratory epithelium and their in vitro cultivation[J]. In Vitro Cell Dev Biol,1997,33 (1):3-14.
2. Uhal BD. Cell cycle kinetics in the alveolar epithelium [J]. Am J Physiol Lung Cell Mol Physiol,1997,272 (6 Pt 1):L1031-L1045.
3. Kinnard W, Tuder R, Papst P, et al. Regulation of alveolar type Ⅱ cell differentiation and proliferation in adult rat lung explants [J]. Am J Respir Cell Mol Biol,1994,11 (4):416-425.
4. Holm BA, Matalon S, Finkelstein JN, et al. Type Ⅱ pneumocyte changes during hyperoxic lung injury and recovery [J]. J Appl Physiol,1988,65 (6):2672-2678.
5. Pison U, Wright JR, Hawgood S. Specific binding of surfactant apoprotein SP-A to rat alveolar macrophages [J]. Am J Physiol Lung Cell Mol Physiol,1992,262 (4 Pt 1):L412-L417.
6. 司徒镇强,吴军正主编.细胞培养.第1版.西安：世界图书出版公司,1996.63-68.
7. Dobbs LG, Geppert EF, Williams MC, et al. Metabolic properties and ultrastructure of alveolar type Ⅱ cells isolated with elastase [J]. Biochim Biophys Acta,1980,618 (3):510-523.
8. Kikkawa Y, Yoneda K. The type Ⅱ epithelial cell of the lung. I. Method of isolation [J]. Lab Invest,1974,30 (1):76-84.
9. Finkelstein JN, Shapiro DL. Isolation of type Ⅱ alveolar epithelial cells using low protease concentrations. Lung,1982,160(2):85-98.
10. Feinstein G, Kupfer A, Sokolovsky M. N-acetyl-(L-Ala) 3-p-nitroanilide as a new chromogenic substrate for elastase. Biochem Biophys Res Commun,1973, 50(4):1020-1026.
11. Sashar LA, Winter KK, Sicher N, et al. Photometric method for estimation of elastase activity. Proc Soc Exp Biol Med,1955,90(2):323-326.
12.曾庆富,蒋海鹰,钱仲等.肺泡Ⅱ型上皮细胞的分离纯化及原代培养.中华病理学杂志,1998,27(5)：384-385.
13.潘芳,李文志.肺泡Ⅱ型上皮细胞的体外培养及其在麻醉学研究领域的应用.国外医学麻醉学与复苏分册.2000,21(6)：368-371.
14. Weller NK, Karnovsky MJ. Improved isolation of rat lung alveolar type Ⅱ cells. More representative recovery and retention of cell polarity. Am J Pathol,1986, 122(1):92-100
15. Murphy S A, Dinsdale D, Hoet P, et al. A comparative study of the isolation of type Ⅱ epit helial cells from rat, hamster, pig and human lung tissue. Methods Cell Sci,1999,21(1):31-38.
16. Kikkawa Y, Yoneda K. The type Ⅱ epithelial cell of the lung. I. Method of isolation. Lab Invest,1974,30:76-84.
17. Bouke K, Boekema H L, Norbert S Z. Adherence of Actinobacillus pleuropneumoniae to primary cultures of porcine lung epithelial cells. Vet Micr, 2003,93:133-144.
18. Funkhouser J D, Cheshire L B, Ferrara T B, et al. Monoclonal antibody identification of a type Ⅱ alveolar epithelial cell antigen and expression of the antigen during lung development. Dev Biol,1987,119:190-198.
19. Rochat T R, Casale J M, Hunninghake G W, et al. Characterization of type Ⅱ alveolar epithelial cells by flow cytometry and fluorescent markers. J Lab Clin Med,1988,112:418-425.
20. Uhal B D, Hess G D, Rannels D E. Density independent isolation of type Ⅱ pneumocytes after partial pneumocytes after partial pneumonectomy. Am J Physiol,1989,256:C515-C521.
21. Dobbs LG, Gonzalez R, Williams MC. An improved method for isolating type Ⅱ cells in high yield and purity [J]. Am Rev Respir Dis,1986,134(1):141-145.
22. Mason RJ, Walker SR, Shields BA, et al. Identification of rat alveolar type Ⅱ epithelial cells with a tannic acid and polychrome stain [J]. Am Rev Respir Dis, 1985,131 (5):786-788.
23. Edelson JD, Shannon JM, Mason RJ. Alkaline phosphatase:a marker of alveolar type Ⅱ cell differentiation [J]. Am Rev Respir Dis,1988,138(5):1268-1275.
24. Diglio CA, Kikkawa Y. The type Ⅱ epithelial cells of the lung. Ⅳ. Adaptation and behavior of isolated type Ⅱ cells in culture [J]. Lab Invest,1977,37 (6):622-631.
25.樊燕蓉,姚汝琳.肺泡Ⅱ型上皮细胞的分离培养及其在矽肺研究中的应用.国外医学卫生学分册,1996,23(4)：212-214.
26. Dobbs LG, Williams MC, Brandt AE. Changes in biochemical characteristics and pattern of lectin binding of alveolar type Ⅱ cells with time in culture. Biochim Biophys Acta,1985,846(1):155-166.
27. Liley HG, Ertsey R, Gonzales LW, et al. Synthesis of surfactant components by cultured type Ⅱcells from human lung. Biochim Biophys Acta,1988,961(1): 86-95.
28. Whitsett JA, Ross G, Weaver T, et al. Glycosylation and secretion of surfactant-associated glycoprotein A. J Biol Chem,1985,260(28):15273-15279
1. Asahara T, Murohara T, Sullivan A, et al. Isolation of Putative Progenitor Endothelial Cells for Angiogenesis. Science,1997,275:964-967.
2. Lam KY, Lo CY, Fan ST. Pancreatic solid-cystic-papillary tumor: Clinicopathologic features in eight patients from Hong Kong and review of the literature [J]. World J Surg,1999,23 (10):1045-1050.
3. Nijs E, Callahan MJ, Taylor GA. Disorders of the pediatric pancreas:Imaging features[J]. Pediatr Radiol,2005,35(4):358-373.
4. Zeytunlu M, Firat O, Nart D, et al. Solid and cystic papillary neoplasms of the pancreas:Report of four cases [J]. Turk J Gastroenterol,2004,15 (3):178-182.
5. Meshikhes AWN, Atassi R. Pancreatic pseudopapillary tumor in a male child [J]. JOP,2004,5(6):505-511.
6. Teleron AA, Carlson B, Young PP. Blood donor white blood cell reduction filters as a source of human peripheral blood-derived endothelial progenitor cells. Transfusion,2005; 45(1):21-5.
7. 肖刚峰,张怀勤,季亢挺等.单个核细胞的接种密度对贴壁选择法培养内皮祖细胞增殖的影响.细胞与分子免疫学杂质,2006,22(2)：233-234
8. Frontczak-Baniewicz M, Gordon-Krajcer W, Walski M. The immature endothelial cell in new vessel formation following surgical injury in rat brain. Neuro Endocrinol Lett,2006,27(4):539-546.
9. Quirici N, Soligo D, Caneva L, et al. Differentiation and expansion of endothelial cells from human bone marrow CD133+cells[J]. Br J Haematol,2001,115(1): 186-194.
10. Hilbe W, Dirnhofer S, Oberwasserlechner F, et al. CD133 positive endothelial progenitor cells contribute to the tumour vasculature in non-small cell lung cancer [J]. Clin Pathol,2004,57(9):965-969.
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2. Uhal BD. Cell cycle kinetics in the alveolar epithelium [J]. Am J Physiol Lung Cell Mol Physiol,1997,272 (6 Pt 1):L1031-L1045.
3. Kinnard W, Tuder R, Papst P, et al. Regulation of alveolar type Ⅱ cell differentiation and proliferation in adult rat lung explants [J]. Am J Respir Cell Mol Biol,1994,11 (4):416-425.
4. Holm BA, Matalon S, Finkelstein JN, et al. Type Ⅱ pneumocyte changes during hyperoxic lung injury and recovery [J]. J Appl Physiol,1988,65 (6):2672-2678.
5. Pison U, Wright JR, Hawgood S. Specific binding of surfactant apoprotein SP-A to rat alveolar macrophages [J]. Am J Physiol Lung Cell Mol Physiol,1992,262 (4 Pt 1):L412-L417.
6. 司徒镇强,吴军正主编.细胞培养.第1版.西安：世界图书出版公司,1996.63-68.
7. Dobbs LG, Geppert EF, Williams MC, et al. Metabolic properties and ultrastructure of alveolar type Ⅱ cells isolated with elastase [J]. Biochim Biophys Acta,1980,618 (3):510-523.
8. Kikkawa Y, Yoneda K. The type Ⅱ epithelial cell of the lung. I. Method of isolation [J]. Lab Invest,1974,30 (1):76-84.
9. Finkelstein JN, Shapiro DL. Isolation of type Ⅱ alveolar epithelial cells using low protease concentrations. Lung,1982,160(2):85-98.
10. Feinstein G, Kupfer A, Sokolovsky M. N-acetyl-(L-Ala) 3-p-nitroanilide as a new chromogenic substrate for elastase. Biochem Biophys Res Commun,1973, 50(4):1020-1026.
11. Sashar LA, Winter KK, Sicher N, et al. Photometric method for estimation of elastase activity. Proc Soc Exp Biol Med,1955,90(2):323-326.
12.曾庆富,蒋海鹰,钱仲等.肺泡Ⅱ型上皮细胞的分离纯化及原代培养.中华病理学杂志,1998,27(5)：384-385.
13.潘芳,李文志.肺泡Ⅱ型上皮细胞的体外培养及其在麻醉学研究领域的应用.国外医学麻醉学与复苏分册.2000,21(6)：368-371.
14. Weller NK, Karnovsky MJ. Improved isolation of rat lung alveolar type Ⅱ cells. More representative recovery and retention of cell polarity. Am J Pathol,1986, 122(1):92-100
15. Murphy S A, Dinsdale D, Hoet P, et al. A comparative study of the isolation of type Ⅱ epit helial cells from rat, hamster, pig and human lung tissue. Methods Cell Sci,1999,21(1):31-38.
16. Kikkawa Y, Yoneda K. The type Ⅱ epithelial cell of the lung. I. Method of isolation. Lab Invest,1974,30:76-84.
17. Bouke K, Boekema H L, Norbert S Z. Adherence of Actinobacillus pleuropneumoniae to primary cultures of porcine lung epithelial cells. Vet Micr, 2003,93:133-144.
18. Funkhouser J D, Cheshire L B, Ferrara T B, et al. Monoclonal antibody identification of a type Ⅱ alveolar epithelial cell antigen and expression of the antigen during lung development. Dev Biol,1987,119:190-198.
19. Rochat T R, Casale J M, Hunninghake G W, et al. Characterization of type Ⅱ alveolar epithelial cells by flow cytometry and fluorescent markers. J Lab Clin Med,1988,112:418-425.
20. Uhal B D, Hess G D, Rannels D E. Density independent isolation of type Ⅱ pneumocytes after partial pneumocytes after partial pneumonectomy. Am J Physiol,1989,256:C515-C521.
21. Dobbs LG, Gonzalez R, Williams MC. An improved method for isolating type Ⅱ cells in high yield and purity [J]. Am Rev Respir Dis,1986,134(1):141-145.
22. Mason RJ, Walker SR, Shields BA, et al. Identification of rat alveolar type Ⅱ epithelial cells with a tannic acid and polychrome stain [J]. Am Rev Respir Dis, 1985,131 (5):786-788.
23. Edelson JD, Shannon JM, Mason RJ. Alkaline phosphatase:a marker of alveolar type Ⅱ cell differentiation [J]. Am Rev Respir Dis,1988,138(5):1268-1275.
24. Diglio CA, Kikkawa Y. The type Ⅱ epithelial cells of the lung. Ⅳ. Adaptation and behavior of isolated type Ⅱ cells in culture [J]. Lab Invest,1977,37 (6):622-631.
25.樊燕蓉,姚汝琳.肺泡Ⅱ型上皮细胞的分离培养及其在矽肺研究中的应用.国外医学卫生学分册,1996,23(4)：212-214.
26. Dobbs LG, Williams MC, Brandt AE. Changes in biochemical characteristics and pattern of lectin binding of alveolar type Ⅱ cells with time in culture. Biochim Biophys Acta,1985,846(1):155-166.
27. Liley HG, Ertsey R, Gonzales LW, et al. Synthesis of surfactant components by cultured type Ⅱcells from human lung. Biochim Biophys Acta,1988,961(1): 86-95.
28. Whitsett JA, Ross G, Weaver T, et al. Glycosylation and secretion of surfactant-associated glycoprotein A. J Biol Chem,1985,260(28):15273-15279
1. Asahara T, Murohara T, Sullivan A, et al. Isolation of Putative Progenitor Endothelial Cells for Angiogenesis. Science,1997,275:964-967.
2. Lam KY, Lo CY, Fan ST. Pancreatic solid-cystic-papillary tumor: Clinicopathologic features in eight patients from Hong Kong and review of the literature [J]. World J Surg,1999,23 (10):1045-1050.
3. Nijs E, Callahan MJ, Taylor GA. Disorders of the pediatric pancreas:Imaging features[J]. Pediatr Radiol,2005,35(4):358-373.
4. Zeytunlu M, Firat O, Nart D, et al. Solid and cystic papillary neoplasms of the pancreas:Report of four cases [J]. Turk J Gastroenterol,2004,15 (3):178-182.
5. Meshikhes AWN, Atassi R. Pancreatic pseudopapillary tumor in a male child [J]. JOP,2004,5(6):505-511.
6. Teleron AA, Carlson B, Young PP. Blood donor white blood cell reduction filters as a source of human peripheral blood-derived endothelial progenitor cells. Transfusion,2005; 45(1):21-5.
7. 肖刚峰,张怀勤,季亢挺等.单个核细胞的接种密度对贴壁选择法培养内皮祖细胞增殖的影响.细胞与分子免疫学杂质,2006,22(2)：233-234
8. Frontczak-Baniewicz M, Gordon-Krajcer W, Walski M. The immature endothelial cell in new vessel formation following surgical injury in rat brain. Neuro Endocrinol Lett,2006,27(4):539-546.
9. Quirici N, Soligo D, Caneva L, et al. Differentiation and expansion of endothelial cells from human bone marrow CD133+cells[J]. Br J Haematol,2001,115(1): 186-194.
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